Polymers are used for drug delivery for a variety of therapeutic purposes. Polymers have also been used in research for the delivery of nucleic acids (polynucleotides and oligonucleotides) to cells with an eventual goal of providing therapeutic processes. Such processes have been termed gene therapy or anti-sense therapy. One of the several methods of nucleic acid delivery to cells is the use of DNA-polycation complexes. It has been shown that cationic proteins like histones and protamines or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular delivery agents.
Preparation of polycation-condensed polyanion (such as DNA) particles is of particular importance for non-viral gene therapy. Optimal transfection activity in vitro and in vivo can require an excess of polycation molecules. However, the presence of a large excess of polycations is potentially toxic to cells and tissues. Moreover, the non-specific binding of cationic particles to cells as well as serum components hinders specific cellular targeting. Positive charge can also have an adverse influence on biodistribution of the complexes in vivo.
Layer-by-layer polymeric assemblies, based on electrostatic interactions of polyanions and polycations, have found applications in different fields of materials science. Such assemblies are fabricated by consecutive deposition of alternating polycation and polyanion layers on a surface of interest by incubation in dilute aqueous solutions of corresponding polyion. However, this method is applicable only to macroscopic substrates since it requires separation of the substrate from excess polyion after addition of each layer. The formation of such assemblies has been demonstrated on planar macrosurfaces [Decher, G., Science, 277, 1232–1237 (1997)] and latex microspheres [Donath, E., Sukhorukov, G B, Caruso, F., Davis, S A, Möhwald, H. Angew. Chem. 110, 2324–2327 (1998)]. This approach is not amenable to fabrication of multilayer coating of smaller particles, such as condensed DNA complexes, due to the problem of separation of the particle from excess polyion after deposition of each layer.
It is known that polycation/polyanion complexes formed in low salt aqueous solution assume morphology of small spherical particles of submicron size [Dautzenberg, H, Hartmann, J, Grunewald, S, Brand, F. Ber. Bunsenges. Phys. Chem. 100, 11024–1032 (1996)]. Recently we have demonstrated that electrostatic complexes <100 nm in diameter can be build around small particles of polycation-condensed DNA [Trubetskoy V S, Loomis A, Hagstrom J E, Budker V G, Wolff J A, Nucleic Acids Res. 27, 3090–3095 (1999); Trubetskoy V S, Slattum P M, Hagstrom J E, Wolff J A, Budker V G. Anal. Biochem. 267:309–313, (1999)]. However, formation of these complexes—without separation from excess polyion during addition of each layer—results in generation of large quantities of blank complexes. The blank particles are formed from interaction of the excess polycation and polyanion and do not contain DNA. Nevertheless, the DNA-containing particles were shown to be useful as gene transfer agents [Prov. Appl. Ser. No. 60/093,153].